The goal of this research is to discern the mechanisms by which physiological signals in the environment within the human host are converted to molecular interactions that govern the expression of virulence genes. The model system to be analyzed is the Vibrio cholerae ToxR virulence regulon, for which a number of parameters that influence gene expression, as well as many of the regulators and target genes, are known and partially characterized. The target virulence genes include the tcp operon, toxT, and other "ToxR activated" genes present on the Vibrio cholerae pathogenicity island, as well as the ctx operon present on a lysogenic bacteriophage. The regulators are encoded by genes distributed around the genome, including toxRS, aphA, aphB, hns, and crp, as well as the tcpPH and toxT genes present on the pathogenicity island. It has recently been determined that multiple regulators function at each target gene promoter. The current proposal focuses on a subset of target promoters and regulators to understand how these regulators function in an interaction with growth condition signals, the promoters, and with each other to control gene expression. In addition, the details of a newly discovered feedback pathway that links pilus biogenesis to virulence gene expression will be investigated. This pathway negatively impacts virulence gene expression when pilus formation is disrupted in certain ways. It may represent a new signal for release from the intestine. Correlating virulence gene expression together with regulatory responses that modulate bacterial physiology represents a new approach that utilizes knowledge of the genome to further our understanding of the basis of virulence gene regulation. These experiments will likely reveal novel virulence factor genes that may prove to be useful vaccine or therapeutic targets. A further understanding of virulence gene expression will also help in the development of ways to modulate it either in vivo or in vitro for improved vaccine production or overproduction of virulence factors for structural analyses. These studies will also help us further understand the mechanism of action of small molecule virulence inhibitors, such as virstatin.